Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.

Center for Molecular and Cellular Bioengineering Biotechnology Center Technische Universität Dresden Dresden Germany

Centre for Drug Discovery and Development Sathyabama Institute of Science and Technology Chennai India

Faculty of Science Charles University Prague Prague Czech Republic

Genome Engineering Laboratory School of Life Sciences University of Westminster London UK

Institute of Molecular Cancer Research University of Zurich Zurich Switzerland

Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

School of Life Sciences University of Bedfordshire Luton UK

School of Medicine University of California San Francisco San Francisco CA USA

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Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation

MutSβ-MutLβ-FANCJ axis mediates the restart of DNA replication after fork stalling at cotranscriptional G4/R-loops